A team of University of Miami investigators requests $4,970,522 to undertake a readily deployable collaborative project that will redefine the proteomics as a context-rich molecular bioinformatics. Proteomics has been hailed as 'the next big thing'after genomics. It has progressed from cataloging the whole complement of proteins, or proteome, to charting their interactions, or interactome. Yet the major predicament in proteomics today is its paucity of in situ contexts. The team proposes to launch an imaging-based survey of protein-protein interaction networks within neurons. Its ultimate goal is reconstruction of genome-wide protein- protein interaction networks within each and every subcellular compartment of neurons at progressive steps of their development. This project will be the first systematic inspection of when and where each protein-protein interaction takes place in vivo. The investigators bring their expertise in neuronal imaging, Drosophila genetics, and computational analysis. The project will isolate GFP (green fluorescent protein) protein-trap lines for endogenous proteins present in well-studied model CNS and PNS neurons, and create inducible transgenic lines for neurologically 'relevant'proteins tagged with either GFP or monomeric RFP (red fluorescent protein). Using crosses to combine the green and red tagged proteins in a single fly, the project will both determine the localization of each protein and reveal the dynamic interactions between proteins in cell body, axon, dendrite and/or synapse of intact neurons within a whole organism during development. To achieve high-content screens within two years, the project takes advantage of a robotic imaging system as well as a FLIM (fluorescent lifetime microscopy) cluster. The latter quantifies GFP-to-RFP FRET (Forster resonance energy transfer), an indicator of direct association, between each of approximately 100,000 protein protein pairs. Over one million 3D images of model neurons will be analyzed to construct proteomic maps specific for different neuronal types, developmental stages and subcellular compartments. This image library will then undergo cross-correlation analysis to arrive at a model of the dynamics of the molecular networks of wild type neurons. At its completion, the project not only delivers the first context-rich proteomics resource, but also offers a new intellectual infrastructure for determining the molecular circuitries affected by neurological disorders, aging or drug addiction and designing strategies to repair and/or protect neurons. PUBLIC HEALTH RELEVANCE: Proteomics remains virtually context-less. The proposed project will show context-specific proteomic maps of wild type neurons in the intact brain, furnishing a much-needed intellectual infrastructure to transform definitions of the molecular circuitries affected by neurological disorders as well as strategies to repair/protect neurons.